Low proportion of BRCA1 and BRCA2 mutations in. Finnish breast cancer families: evidence for additional susceptibility genes. Paula Vehmanen+, Lori S.
1997 Oxford University Press
Human Molecular Genetics, 1997, Vol. 6, No. 13 2309–2315
Low proportion of BRCA1 and BRCA2 mutations in Finnish breast cancer families: evidence for additional susceptibility genes Paula Vehmanen+, Lori S. Friedman1,+, Hannaleena Eerola2, Melody McClure3, Brian Ward3, Laura Sarantaus, Tommi Kainu4, Kirsi Syrjäkoski4, Seppo Pyrhönen2, Olli-P. Kallioniemi4, Timo Muhonen2, Michael Luce3, Thomas S. Frank3 and Heli Nevanlinna* Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Haartmaninkatu 2, 00290 Helsinki, Finland, 1CRC Human Cancer Genetics Group, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QQ, UK, 2Department of Oncology, Helsinki University Central Hospital, Haartmaninkatu 4, 00290 Helsinki, Finland, 3Myriad Genetic Laboratories, 390 Wakara Way, Salt Lake City, UT 84108, USA and 4Laboratory of Cancer Genetics, Institute of Medical Technology, Tampere University Hospital, Lenkkeilijänkatu 6, 33520 Tampere, Finland Received August 21, 1997; Revised and Accepted September 24, 1997
One hundred breast and breast-ovarian cancer families identified at the Helsinki University Central Hospital in southern Finland and previously screened for mutations in the BRCA2 gene were now analyzed for mutations in the BRCA1 gene. The coding region and splice boundaries of BRCA1 were analyzed by protein truncation test (PTT) and heteroduplex analysis (HA)/SSCP in all 100 families, and 70 were also screened by direct sequencing. Contrary to expectations based on Finnish population history and strong founder effects in several monogenic diseases in Finland, a wide spectrum of BRCA1 and BRCA2 mutations was found. In the BRCA1 gene, 10 different protein truncating mutations were found each in one family. Six of these are novel Finnish mutations and four have been previously found in other European populations. Six different BRCA2 mutations were found in 11 families. Altogether only 21% of the breast cancer families were accounted for by mutations in these two genes. Linkage to both chromosome 17q21 (BRCA1) and 13q12 (BRCA2) was also excluded in a subset of seven mutation-negative families with four or more cases of breast or ovarian cancer. These data indicate that additional breast and breast-ovarian cancer susceptibility genes are likely to be important in Finland. INTRODUCTION The most prominent risk factor for breast cancer is a family history. It is estimated that 5–10% of all breast cancers may arise from hereditary predisposition (1,2). The two currently known
major breast cancer predisposing genes, BRCA1 (3,4) and BRCA2 (5–7) were originally thought to account for the vast majority of breast cancer families. BRCA1 was reported to account for ∼45% of hereditary breast cancer families (especially those with ovarian cancer), and BRCA2 for an additional 35%, (including those with male breast cancer) (5,8). These estimates were largely based on studies of the same large families that were initially used to assign linkage to these two genes. Very recently, large scale mutation analyses of these two genes indicate that in many populations only ∼30–60% of breast cancer families are attributable to BRCA1 and BRCA2 mutations. The proportion of large breast cancer pedigrees that are attributable to BRCA1 and BRCA2 is 21 and 9% in Britain, 24 and 18% in France, 40 and 16% in Canada, and 39 and 25% in the USA, respectively (9). In Sweden and in Hungary, ∼35% of families carry mutations in these two genes (9). In isolated populations with a strong founder effect, the situation may be different. In Iceland, a single BRCA2 founder mutation accounts for the majority of hereditary breast cancers (10,11), and in the Ashkenazi Jewish population, a very high carrier frequency has been reported for the three founder mutations, 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 (12–14). The Finnish population is also considered an isolate. There are strong founder effects for several characteristic Finnish genetic diseases, while several genetic diseases that are common elsewhere in the world, are rare or even unknown in Finland (15). Therefore, substantial interest in the BRCA1 and BRCA2 mutation spectrum and its significance in this country exists. We recently reported the analysis of the BRCA2 mutation spectrum in 100 Finnish breast cancer families with three or more breast or ovarian cancers (16). We have now analyzed mutations of the BRCA1 gene in the same families, to complete the survey of the spectrum of mutations predisposing to breast cancer in the Finnish families. Additional data on recurrent BRCA2 mutations were also obtained. The mutation
% !!" " # !"" & $ "i +These authors contributed equally to the study
2310 Human Molecular Genetics, 1997, Vol. 6, No. 13 spectrum was surprisingly wide. Ten different BRCA1 mutations and six BRCA2 mutations were discovered. Although several of these mutations were recurrent, there was no evidence of a major founding mutation in either gene. Only 21% of breast cancer families could be attributed to the effects of these two genes, suggesting that additional breast cancer predisposition genes are likely to be important in Finland. RESULTS AND DISCUSSION BRCA1 mutations Mutation analysis of the BRCA1 gene coding region and splice boundaries in 100 index cases by protein truncation test (PTT) and heteroduplex analysis (HA)/SSCP analysis, as well as by
direct sequencing in 70 of these, revealed 10 different mutations each in one family (10%) (95% CI = 5–18) (Table 1). All of the mutations segregated with cancer in the families. Six of the 10 mutations were frameshift mutations including five deletions and one insertion, and three were base substitutions leading to immediate termination codon. One mutation was an exon 12 splice acceptor site nt –2 A→G substitution (Table 1). This mutation destroys the nearly invariant AG of the splice acceptor site (17) and is predicted to cause aberrant splicing and truncation of the protein product. This splice substitution was not seen in 186 normal control chromosomes as determined by ASO hybridization, and we therefore classified it as a mutation. The mutations were unevenly located over the BRCA1 gene, with five mutations found in exon 11 and five mutations in exons 12–20.
Table 1. BRCA1 mutations and phenotypes in the familiesa Family
Mutation
Status
Breast cancer (ages of diagnosis, years)
Ovarian cancer
Breast and ovary
254
Exon 11 1924 delA
Finnish
2 (44,58)
1 (16)
skin (47)
160
Exon 11 2803 delAA
Dutch founder
2 (51,72)
2 (52,70)
unknown
112
Exon 11 3604 delA
Dutch/German
0
2 (43,48)
188
Exon 11 3744 delT
Finnish Recurrent (3)
1 (bil) (30/42)
1 (48)
167
Exon 11 3904 C→A
Finnish
2 (39,55)
2 (66, na)
136
Exon 12 nt –2 A→G
Finnish Recurrent (2)
3 (1 bil) (28,34,43/51)
0
286
Exon 13 4446 C→T
Hot spot? Recurrent (2)
9 (25,32,36,41,44,55,55,71,na)
0
lung (na)
141
Exon 17 5145 del11
Finnish
3 (32,35,35)
0
peritoneal metastasis (female), primary unknown (46), gall bladder (56)
263
Exon 20 5370 C→T
Finnish Recurrent (2)
2 (55,59)
280
Exon 20 5385 insC
N-E Europe/ Jewish founder
1 (49)
1 (41,44)
lung cancer (76)
stomach (2 cases) skin (55) 1 (52,50)
1 (50,57)
1 (na)
Other cancers
peritoneal metastasis (female), primary unknown (58) skin (43), corpus carcinoma (61), pancreas (74), lung (63), salivary gland (87)
peritoneal metastasis (female), primary unknown (d61), skin (63,74) lymphoma (69), multiple myeloma (80) skin (78)
Total: 28 patients with breast cancer (two bilateral), median age of onset 43.9 years, (25 patients with breast cancer only, three with breast and ovarian cancer). Twelve patients with ovarian cancer, median age of onset 49 years (nine patients with ovarian cancer only, three with breast and ovarian cancer). aCarrier status of all family members is not known. na, not available.
2311 Human Genetics, 1997, 6, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 13 2311 exon 24 splice acceptor mutation (9346 nt –2 A→G) have now each been found in five unrelated families. Accumulation of BRCA1 and BRCA2 mutation data from larger sets of families as well as unselected breast and ovarian cancer patients will reveal the prevalence of the different mutations and the significance of the recurrent, putative founder mutations in Finland. The large number of different BRCA1 and BRCA2 mutations identified in this study, and low frequency of any recurrent mutation so far, suggest that there may not be a strong BRCA1 or BRCA2 founder in Finland. Figure 1. PTT analysis, revealing the 3904 C→A mutation in family 167 (A) and the 3604delA mutation in family 112 (B).
Missense variants of unknown consequence and infrequent polymorphisms were also found and are deposited in the BIC data base (18). Recurrent BRCA1 and BRCA2 mutations In this population of 100 families, each of the ten BRCA1 mutations were found in one family only. However, outside this cohort we have identified four of the mutations in additional Finnish families by ASO hybridization (Table 1). The exon 11 nt 4446 C→T substitution, the exon 12 splice acceptor mutation at nucleotide –2 A→G and the exon 20 nt 5370 C→T substitution were identified each in one additional family. The exon 11 mutation 3744delT was additionally identified in a breast-ovarian cancer family and in a third family with three sisters affected with ovarian cancer, from a set of ovarian cancer families described elsewhere (19). Thus four of the mutations found here were recurrent, three were found in two families and one was found in three families. This cohort of 100 breast cancer families was previously found to carry five different BRCA2 mutations in eight families (8%) by PTT and HA/SSCP analysis (16). In the present study, linkage analysis of 12 families (see below) revealed haplotype sharing in chromosome 13q12 in one breast-ovarian cancer family. Direct sequencing of the BRCA2 gene in the index patient DNA sample revealed a protein truncating mutation, 7708 C→T substitution in exon 15. This mutation was not detected by HA/SSCP analysis in the previous study, and has not been reported previously. ASO-hybridization analysis of the 100 index case samples identified two other families with this mutation in this cohort. In summary, a total of six different BRCA2 mutations has now been detected in 11 families (11%) (95% CI = 6–19) from the cohort of 100 families. Outside this cohort, the 7708 C→T mutation was also found in one additional family by ASO hybridization. Thus, four unrelated families carry the 7708 C→T mutation. The ancestry of all four families originates from the south-eastern part of the country, as dated back to the 1800s, suggesting a regional founder mutation. Previously, we described three recurrent BRCA2 mutations as possible founding mutations each in two Finnish families (16). One of these was the Icelandic founder mutation 999del5. In follow-up studies of other families, we have now identified this mutation in seven unrelated Finnish families (including two from a set of breast cancer families described elsewhere; Ralf Krahe and Juha Kere, personal communication). The other recurrent mutations, the exon 18 nonsense mutation (8555T→G) and the
Finnish–European mutations Of the 10 BRCA1 mutations identified here, six are so far novel Finnish mutations while four have been described previously in other European populations or in the USA (Table 1). Of these, 2803delAA (also called 2804delAA) is a prominent founder mutation in The Netherlands and Belgium, but has not been found previously in other populations (20). Another mutation, 3604delA (Fig. 1), has been described in Dutch and German families (18,20). The 5385insC (also named as 5382insC) mutation found in one Finnish family has been described as a founder mutation in Russia (21) and in Hungary (22), in families of Jewish and non-Jewish ancestry. Finally, the 4446C→T mutation in exon 13 has been found multiple times on different haplotypes and may represent a mutational hotspot (23). Of the six different BRCA2 mutations, five were novel mutations so far unique to Finland, while one recurrent, and a proposed ancient founder mutation (999del5) (16) has been previously described as a strong founder in Iceland (10,11). It is of interest that the 999del5 mutation is also the most frequently found mutation in Finland so far. While some of the mutations described in other European populations may have arrived in Finland during the past centuries, or have an independent origin, others, especially the 999del5 mutation, may reflect ancient genetic relationships between European populations (16). Additional haplotype analysis of the Finnish families carrying the recurrent mutations, including both the country-specific and Finnish–European mutations, will be required to establish the nature of these putative founder mutations. Haplotypes around shared mutations between the Finnish and other European families may help to estimate the age of these mutations as well as explain their emergence in Finland. Phenotype in BRCA1 mutation families The ages at diagnosis, and the number and types of cancer in each family are shown in Table 1. An early age of breast cancer onset is clearly an indicator of BRCA1 as well as BRCA2 mutation carrier status in these Finnish breast cancer families. The age of onset in the BRCA1 (median 43.9 years) (P
